The project concerns the molecular biology of yellow fever virus, the prototype of a group of medically important viruses which includes many pathogens causing disease in humans worldwide. The replication of these positive-strand RNA viruses involves synthesis of a large polyprotein and processing by a specific virus-encoded protease. The specific objectives of the proposed studies are to identify and characterize the viral protease(s) responsible for polyprotein processing, including large scale purification for biochemical and structural analysis. The methods employed for this involve deriving in vitro systems for detailed analysis of the protease activity, including the identification and site-directed mutagenesis of the active site, characterization of cleavage specificity toward a variety of native and altered substrates, and in vivo characterization of relevant protease mutations using a yellow fever infectious clone. It is anticipated that these studies will broaden understanding of proteases as components of viral pathogenesis, and lead towards rational design of antiviral drug therapy.